Project Summary/Abstract The overarching objective of this proposal is to develop a robust approach to map the brain's connections quickly, accurately, and cost-effectively. Past efforts to address the challenge of teasing apart the complex connectome of the mammalian brain were subject to a steep trade-off between throughput/efficiency and resolution. Two cutting-edge neuronal mapping techniques?barcoding based connection mapping (BARseq) and expansion microscopy (ExM)?have proven they can achieve efficient and high-resolution connection mapping within mammalian neural tissue. We will optimize and then integrate these two techniques to map both local and long-range circuitry with a single-synapse resolution. In ExM, neural tissue is physically expanded, making it easier to disambiguate neural fibers in close proximity and to detect the precise location of synapse-associated proteins. This approach is ideally suited to teasing apart the paths and connections among densely packed local circuits. Using BARseq, neurons express unique barcoded tags, which allows even distant processes to be accurately traced to their somatic origins. Combining BARseq with in situ immunolabeling techniques, we can also precisely identify the location of synapses on each fiber. Here we propose to optimize the combination of these two approaches, which will enable a platform for generating a brainwide microconnectome with single-synapse level resolution. Success in this effort has clear implications for the future of neuroscience research, including the potential to transform our understanding of both normal brain circuitry and the specific disruptions that occur within the context of neuropsychiatric disorders.